Abstract

Calcineurin (CN), a calcium- and calmodulin-dependent protein phosphatase,
plays a significant role in the central nervous system. Previously, we
reported that forebrain-specific CN knockout mice (CN mutant mice) have
impaired working memory. To further analyze the behavioral effects of CN
deficiency, we subjected CN mutant mice to a comprehensive behavioral test
battery. Mutant mice showed increased locomotor activity, decreased social
interaction, and impairments in prepulse inhibition and latent inhibition. In
addition, CN mutant mice displayed an increased response to the locomotor
stimulating effects of MK-801. Collectively, the abnormalities of CN mutant
mice are strikingly similar to those described for schizophrenia. We propose
that alterations affecting CN signaling could comprise a contributing factor
in schizophrenia pathogenesis.

Calcineurin (CN), also called protein phosphatase 2B, is a heterodimeric
Ca2+/calmodulin-dependent serine/threonine protein phosphatase
composed of CNB regulatory and CNA catalytic subunits
(1). Originally identified in
the brain, CN was later found to play a critical role in T cell function,
through activation of nuclear factor of activated T cell-mediated
transcription of cytokine genes, including the IL-2 gene
(2,
3). This action of CN comprises
a target for the immunosuppressants, cyclosporin A and FK506, which associate
with immunophilins and bind to and inactivate CN. More recently, CN has been
shown to play an important role in CNS functions, including neurite extension,
synaptic plasticity and learning and memory
(4,
5).

We previously reported a severe and specific working memory deficit of
forebrain specific CNB-deficient mice (CN mutant mice) as assessed by delayed
matching to place Morris water maze and eight-arm radial maze paradigms
(5). To further investigate the
behavioral significance of CN, CN mutant mice were subjected to a
comprehensive behavioral test battery
(6,
7). CN mutant mice display a
spectrum of abnormalities that is strikingly similar to those observed in
schizophrenia patients. In addition, a number of supporting lines of evidence
are consistent with the possibility that alterations in CN function occur in
schizophrenia. Furthermore, in our accompanying paper, we investigated the
potential involvement of altered CN signaling in genetic susceptibility to
schizophrenia, and we report evidence supporting association of the PPP3CC
gene encoding the CNAγ catalytic subunit with disease
(8). Based on these findings,
we propose that alterations affecting CN signaling could comprise an important
contributing factor in schizophrenia pathogenesis.

Materials and Methods

Animals and Experimental Design. The generation of the CN mutants is
detailed elsewhere (5). The
background strain used to generate the mutation was C57BL/6J. All mutant and
control mice were in a pure C57BL/6J background. All CN mutant mice consisted
of homozygous floxed, heterozygous Cre recombinase transgenic mice. All
control mice consisted of homozygous or heterozygous floxed, Cre transgene
negative littermates. All behavioral tests were carried out with male mice
that were 10 weeks old at the start of the testing. Mice were housed in a room
with a 12-hr light/dark cycle (lights on at 7:00 a.m.) with access to food and
water ad libitum. Behavioral testing was performed between 9:00 a.m. and 5:00
p.m. except for the home cage social interaction test. All procedures relating
to animal care and treatment conformed to Massachusetts Institute of
Technology and National Institutes of Health guidelines. Sequences of tests,
housing conditions, and the number of animals used are shown in Table 1, which
is published as supporting information on the PNAS web site,
www.pnas.org.
The methods for rotarod, hot plate, light/dark transition, elevated plus maze,
object exploration, social interaction (in a novel environment), Porsolt
forced swim tests, in vivo microdialysis, and HPLC assessment of
brain content of monoamines and metabolites are described in Supporting
Text, which is published as supporting information on the PNAS web
site.

Open Field Test. Each subject was placed in the center of an open
field apparatus (40 × 40 × 30 cm; Accuscan Instruments, Columbus,
OH). Total distance traveled (in cm), vertical activity, time spent in the
center, and the beam–break counts for stereotyped behaviors were
recorded. Data were collected over a 60-min period.

Social Interaction in Home Cage. To monitor social behavior between
two mice in a familiar environment, a system that automatically analyzes
social behavior in home cages of mice was developed (Fig. 7, which is
published as supporting information on the PNAS web site). The system contains
a home cage (29 × 18 × 12 cm) and a filtered cage top, separated
by a 13-cm-high metal stand containing an infrared video camera, which is
attached at the top of the stand. Two genetically identical mice that had been
housed separately were placed together in a home cage. Their social behavior
was then monitored for 3 days. Outputs from the video cameras were fed into a
Macintosh computer. Images from each cage were captured at a rate of one frame
per second. Social interaction was measured by counting the number of
particles in each frame: two particles indicated the mice were not in contact
with each other; and one particle demonstrated contact between the two mice.
We also measured locomotor activity during these experiments by quantifying
the number of pixels changed between each pair of successive frames. For
details of the image analysis, see Supporting Text.

Latent Inhibition Test. This experiment was performed in a similar
manner to that reported by others
(9) with the equipment used by
Zeng et al. (5). On
the first day, each mouse was placed in a conditioning chamber (Coulbourn
Instruments, Allentown, PA). The mice were divided into two groups: preexposed
(P) group and non-preexposed (NP) group. The P group received 40 white noise
tones (68 dB, 5-sec duration, 25-sec interstimulus interval), whereas the NP
group received no stimulus during an equivalent period. Immediately after the
tone preexposure or the exposure to the chamber, tone–shock pairs
consisting of a 5-sec tone coterminating with a 2-sec foot shock at 0.40 mA
were delivered to both groups with a 25-sec interstimulus interval. Afterward,
mice remained in the chamber for 25 sec before being returned to the home
cage. On day 2, the mice were placed back in the conditioning chamber for 5
min for the measurement of freezing to the context. On day 3, the mice were
put in a white Plexiglas chamber scented with vanilla essence, and after 180
sec, a 180-sec tone was delivered to measure cued freezing.

Prepulse Inhibition Task. A startle reflex measurement system was
used (MED Associates, St. Albans, VT) and the experiment was performed in a
manner identical to that of Miyakawa et al.
(7). See Supporting
Text for details.

Sensitivity to MK-801 and Amphetamine. To test the locomotor
activating effects of MK-801 or amphetamine (Sigma), mice were habituated to
an open field for 1 h, and then drugs were administered i.p. Amphetamine or
MK-801 were dissolved in saline and administered at 0.1 ml per 10 g of body
weight. Because there was a difference in baseline locomotor activity between
genotypes, the ratio of activity during the hour after injection to the
activity during the hour before injection was used as an index of the
locomotor activating effects of the drugs.

Quantification of Nesting. One piece of nesting material (Nestlets,
Ancare, Bellmore, NY), made of cotton fiber, was introduced into a cage in
which a mouse was individually housed. Pictures of the nests were taken by a
digital camera (Olympus, Melville, NY) and exported into a computer. The
number of scattered particles of the nestlets was counted for each cage by the
NIH image program.

In Vivo Microdialysis.In vivo microdialysis measurements
of extracellular dopamine and metabolites were performed in freely moving mice
in an identical manner to that described in Mohn et al.
(10). See Supporting
Text for details.

Image Analysis. All applications used for the behavioral studies
(Image SI, Image OE, Image LD4, Image PS, Image OF, Image HA, and Image FZ)
were run on Macintosh computers. Applications were based on the public domain
NIH image program (developed by Wayne Rasband at the National
Institute of Mental Health, Bethesda) and were modified for each test by
Tsuyoshi Miyakawa (7)
(available through O'Hara & Co., Tokyo).

Results

General Characteristics. CN mutants weighed ≈12% less than their
wild-type littermates (controls, 30.3 ± 0.7; CN mutants, 26.6 ±
0.7; P < 0.01). There were no significant differences in motor
coordination (accelerating rotarod test) and pain sensitivity (hot plate test)
(Fig. 8 A and B, respectively, which is published as
supporting information on the PNAS web site). Although CN mutant mice appear
grossly normal and healthy, they are prone to sudden and acute deterioration
of health leading to death within several days of onset, such that ≈50% of
CN mutants die by 6 months of age. Although we cannot formally exclude a
possible relation between this health effect and the behavioral abnormalities
(see below), we do not believe it is a major contributing factor for several
reasons. First, the onset of this deterioration is sudden and obvious, and
therefore, affected mice were excluded from behavioral testing. Second, as
previously reported, CN mutant mice perform normally in a number of behavioral
tasks including the hidden platform version of the Morris water maze and cued
and contextual fear conditioning
(5). Thus, it is unlikely that
specific behavioral abnormalities in these mice are a reflection of general
health effects.

Increased Locomotor Activity. CN mutants consistently showed a
pronounced increase in locomotor activity in several different tests. Total
distance traveled by CN mutants was significantly greater than that of
controls during an open field test (Fig.
1A; P < 0.0001), elevated plus maze test
(Fig. 9E, which is published as supporting information on the PNAS
web site; P < 0.0001), object exploration test (Fig. 10A,
which is published as supporting information on the PNAS web site), social
interaction test (Fig. 11A, which is published as supporting
information on the PNAS web site), and in the home cage
(Fig. 2B, P =
0.0082). The number of vertical activities in the open field test
(Fig. 1B) and the
stereotypy counts (Fig.
1C) were also significantly increased in the CN mutants
relative to controls (P = 0.010, P = 0.001,
respectively).

Increased locomotor activity of CN mutant mice in the open field test.
Distance traveled (cm) (A), vertical activity (B), and
stereotypic behavior counts (C) were significantly greater in CN
mutants than in controls. Time spent in center (D) was shorter during
the first 30 min in CN mutants relative to controls.

Decreased social interaction in home cage. Number of particles in home cage
(A), an index of social activity, and activity level (B) are
shown. (C and D) All frames taken during an hour were
averaged. Control and CN mutants were active during the dark cycle
(C; 11:00 p.m. to 12:00 p.m.). During the light cycle (D;
11:00 a.m. to 12:00 a.m.), control and CN mutants were inactive. During
inactive periods, control mice stayed in contact with their cage mates,
whereas mutant mice tended to stay separated from each other.

During the Porsolt forced swim test, there were no significant differences
between genotypes in the distance traveled, although the time spent in
immobile posture was significantly shorter in CN mutants than in controls
(Fig. 10C), which may reflect general hyperactivity of CN mutants.
The increase in activity or flight behavior of CN mutant mice complicated the
assessment of their anxiety-like behavior (see Fig. 9 and Supporting
Text for a discussion of abnormal anxiety-like behavior of CN mutant
mice).

Decreased Social Interaction. During a 10-min social interaction
test in a novel environment, the number of contacts between CN mutants did not
differ significantly from that of control mice, except for the first minute,
during which mutant mice made less contacts than controls (Fig. 11B;
genotype × time interaction, P = 0.0031; genotype effect during
the first minute, P = 0.0008). The total duration of contacts and the
mean duration per contact of CN mutants were significantly shorter than those
of control mice (Fig. 11 D and C; P = 0.0088 and
P = 0.0005, respectively). However, because locomotor activity of the
CN mice was increased in this test, it is possible that their decreased
duration of contacts was a consequence of increased locomotion. To confirm
that the decrease in social interaction was not simply the result of
hyperactivity in CN mutants, we monitored social interaction in the home cage
over a 3-day period. Overall, the time that CN mutants spent separated from
each other was significantly greater than that of controls
(Fig. 2A, P
< 0.0001). In fact, even during the light cycle, when mice are usually
sleeping, the time spent in separation was significantly greater in CN mutants
compared with controls (Fig. 2 A,
C, and D; P < 0.0001, P =
0.0037, and P = 0.0073 for light phase of day 2, day 3, and day 4,
respectively). Overall, locomotor activity was also significantly greater in
CN mutants (Fig.
2B).

Impaired Prepulse Inhibition. The startle amplitudes were not
significantly different between genotypes at “startle stimulus
only” trials for any stimulus amplitudes
(Fig. 3A). The percent
prepulse inhibition, an index of sensorimotor gating, was significantly lower
in CN mutants than in controls (Fig.
3B; overall ANOVA including all four conditions,
P = 0.0015). The difference between genotypes was greater when the
startle stimulus intensity was 110 dB (P < 0.0001). At the
stimulus intensity of 120 dB, a significant difference between genotypes was
not observed (P = 0.0922), probably because of a ceiling effect
caused by the strong intensity of the startle stimulus.

Impaired prepulse inhibition (A and B) and latent
inhibition (C–F) of CN mutant mice. Startle amplitude
was not different between genotypes (A), but the percentage prepulse
inhibition was significantly smaller in CN mutants compared with controls
(B). Percent freezing during the conditioning phase was significantly
less in CN mutants (D) compared with controls (C), most
likely due to their hyperactivity. CN mutants traveled longer distances in
response to shocks than controls (Insets in D and
C, respectively). Freezing during contextual testing was not
significantly different between genotypes (C and D). In cued
testing, the percent freezing for the P group was significantly lower than
that of the NP group in control mice, indicating significant latent inhibition
in control mice (E). In contrast, the CN mutants failed to show a
significant latent inhibition (F).

Impaired Latent Inhibition. Percent immobility during the preshock
period of the conditioning phase was significantly lower in mutant mice than
in controls (Fig. 3 C and
D; P = 0.0030), reflecting their hyperactivity.
The percent freezing during the postshock period of the conditioning trial and
during contextual testing were not statistically different between genotypes
(P = 0.4721 and P = 0.6419, respectively). The distance
traveled during shock presentations was significantly greater in the CN
mutants compared with controls (Fig. 3
C and DInsets; P <
0.0001), probably because of their increased locomotor activity and flight
behavior. In cued testing, the percent freezing for the P group was
significantly lower than that of the NP group
(Fig. 3E; P =
0.010) in control mice, indicating significant latent inhibition in control
mice. In contrast, the CN mutants failed to show a significant latent
inhibition (Fig.
3F, P = 0.9248). No significant difference in
freezing between genotypes was observed in the pretone period of cued
testing.

Impaired Nesting Behavior. We noticed, by casual observation, that
nests of CN mutants were poorly formed. Normal mice usually form a clean and
identifiable nest in a distinct location in the cage
(Fig. 4 A and
B). However, the CN mutants did not generally form
distinguishable discrete nests and tended to scatter pieces of nesting
material over the floor of the cage (Fig. 4
C and D). Therefore, pictures of the nests were
taken and the number of scattered particles of nesting material was counted
for each cage. The number of particles in the cages of CN mutants was
significantly larger than that of controls (controls: 9.5 ± 2.3, CN
mutants: 18.2 ± 3.2; P = 0.037).

Impaired nesting behavior. Representative pictures of the cages of control
mice (A and B) and CN mutant mice (C and
D).

Enhanced Sensitivity to the Locomotor Stimulatory Effect of MK-801.
CN is activated after N-methyl-d-aspartate (NMDA) receptor
activation and is a downstream element in dopaminergic signaling. To
investigate the effects of NMDA receptor antagonism and dopamine receptor
activation in the CN mutant mice, we assessed the locomotor stimulatory
effects of a NMDA receptor blocker, MK-801, and an indirect dopaminergic
agonist, amphetamine. CN mutants showed dramatically enhanced locomotor
stimulatory effects of MK-801, a competitive blocker of the NMDA receptor
(Fig. 5 C, D, and
F; P < 0.0001 compared with control mice on
ratio index). Interestingly, the dose response of MK-801 seems to be shifted,
because 0.1 mg/kg of this drug did not affect wild-type mice, but potently
activated mutant mice. In contrast, the locomotor stimulatory effects of
amphetamine did not seem to differ between genotypes
(Fig. 5 A, B, and
E). Although CN mutants were “less sensitive”
in terms of ratio index, this apparent decrease of ratio might be due to the
increased baseline locomotor activity of CN mutants.

Locomotor activating effects of amphetamine and MK-801. Locomotor
stimulation after amphetamine injection was observed in both genotypes
(A and B), although CN mutants were less sensitive to
amphetamine in terms of the ratio between activity before injection and
activity after injection (E). On the other hand, the locomotor
stimulatory effect of MK-801 was significantly greater in CN mutants compared
with controls (C, D, and F; 7–10 animals were used for
each group).

Normal Dopamine Release and Metabolism. Locomotor hyperactivity is
typically associated with increased dopaminergic transmission in major motor
brain areas such as the striatum. To assess the impact of CN mutation on the
state of dopaminergic transmission, a detailed neurochemical analysis was
performed. HPLC analysis of dopamine, serotonin, and metabolites did not
detect any alterations in their tissue levels in the striatum or frontal
cortex of mutant mice (Fig. 12, which is published as supporting information
on the PNAS web site). Similarly, no difference in frontal cortex
norepinephrine levels was observed (Fig. 12). Furthermore, a low perfusion
rate quantitative microdialysis in freely moving mice did not reveal any
significant difference in striatal extracellular levels of dopamine or its
metabolites DOPAC and HVA (Fig.
6). Thus, locomotor hyperactivity and other aberrant behaviors in
CN mutant mice are not associated with observable alterations in monoaminergic
transmission.

Discussion

We have performed a comprehensive behavioral analysis of forebrain-specific
CN mutant mice. These mice were found to have increased locomotor activity,
decreased social interaction, impaired attentional function as assessed by
prepulse (PPI) inhibition and latent inhibition (LI) tests and impaired
nesting behavior. Previously, we reported a severe working memory deficit in
these mutant mice (5). In
addition to these innate behavioral abnormalities, CN mutant mice displayed
increased sensitivity to the locomotor stimulatory effects of MK-801.
Impairments in working memory
(11,
12), PPI
(13), LI
(14), and social withdrawal
(15) are prominent features of
schizophrenia symptomatology. Hyperactivity is characteristic of rodent models
of schizophrenia (16) and
could correspond to psychomotor agitation present in schizophrenic patients.
Furthermore, symptoms of schizophrenic patients are exacerbated by drugs with
NMDA receptor antagonistic properties
(17). Thus, the spectrum of
abnormalities in CN mutant mice is strikingly similar to that observed in
schizophrenia patients. These findings identify the CN mutant mice as a novel
schizophrenia mouse model, and suggest that alterations in CN function could
contribute to schizophrenia etiology.

Other Rodent Models. The behavioral abnormalities of the CN mutant
mice are similar to those observed in a number of currently available rodent
models that recapitulate certain aspects of schizophrenia, such as animals
injected with indirect dopamine agonists
(18,
19), dopamine transporter
knockout and knockdown mice
(16,
20), mice lacking Dvl1
(21), NMDA receptor knockdown
mice (10), and mice carrying
targeted point mutations in the glycine binding site of NMDA receptors
(22). The CN mutant mice
differ from other models in the regionally restricted nature of the lesion. In
CN mutant mice, the gene encoding CNB is disrupted specifically in the cortex,
hippocampal formation and amygdala, whereas expression in the basal ganglia,
including the striatum, is intact. It is notable that this limited ablation
leads to such a comprehensive spectrum of behavioral abnormalities, and
supports the idea that higher brain regions such as the cortex could comprise
a primary site of dysfunction in schizophrenia pathogenesis. Consistent with
this notion, rats with neonatal hippocampal lesions have also been considered
to comprise a rodent schizophrenia model
(19,
23).

Relation to Glutamatergic and Dopaminergic Mechanisms. Our
electrophysiological analysis of CN mutant mice has so far been limited to the
hippocampus (5). In that study
we found a deficiency in NMDA receptor-dependent long-term depression with a
consequent decrease in the range of bidirectional synaptic modifiabilty. It is
of interest to extend this analysis to determine whether similar
electrophysiological deficits occur in the cortex. A major explanatory
hypothesis implicating glutamatergic dysfunction in schizophrenia pathogenesis
is based on the observation that compounds with NMDA-receptor antagonistic
properties evoke behavioral abnormalities similar to those observed in
schizophrenia patients (17).
The nature of affected pathways downstream of the NMDA receptor remains to be
determined. Our findings raise the interesting possibility that alterations in
NMDA-receptor-dependent, CN-mediated phosphatase signaling could be of
particular relevance.

Another explanatory hypothesis proposes that alterations in dopaminergic
signaling comprise a major contributing factor in schizophrenia pathogenesis
(18). More recently, this
concept has evolved to propose that decreased cortical and increased
mesolimbic dopaminergic transmission could account for various cognitive and
behavioral manifestations of schizophrenia
(24). One key function of CN
is dephosphorylation of DARPP-32/inhibitor 1 leading to activation of protein
phosphatase 1 (25). Because
DARPP-32 phosphorylation is a consequence of dopamine D1 receptor activation,
CN could in this way act as a brake in D1-mediated signaling. Thus, although
striatal dopaminergic tone is normal in CN mutant mice, absence of CN activity
in the amygdala, hippocampus, and entorhinal cortex could recapitulate some
aspects of increased mesolimbic dopaminergic transmission. Moreover, because
CN is activated by dopamine D2 receptor signaling
(25), absence of CN could
mimic certain aspects of decreased D2-mediated dopaminergic transmission.
Because CN is involved in NMDA and dopamine receptor signaling pathways, its
absence could disrupt critical interactions between the glutamatergic and
dopaminergic neurotransmitter systems.

Normal Striatal Monoamine Levels and Response to Amphetamine.
Although evidence for increased dopaminergic transmission in schizophrenia is
not conclusive (26), several
studies have detected increased striatal dopaminergic transmission in
schizophrenia patients
(27–29).
Our analysis of monoamine levels revealed normal levels of dopamine and its
metabolites in the striatum of CN mutant mice. There are several explanations
for this possible inconsistency with schizophrenia pathophysiology. First,
because schizophrenia is a complex genetic disease, it is unlikely that all
behavioral and biochemical aspects would be recapitulated by a single genetic
modification. For example, NMDA receptor knockdown mice also display a number
of rodent correlates of schizophrenia symptomatology, and yet have normal
striatal dopaminergic transmission
(10). Second, unlike naturally
occurring genetic variations, the mutation in these mice is restricted to the
cortex, hippocampus, and amygdala, leaving CNB expression in the basal ganglia
intact. It is possible that deletion of CNB in the striatum or elsewhere in
the basal ganglia, particularly in the substantia nigra or ventral tegmentum,
could lead to increased dopaminergic transmission or sensitivity to
amphetamine. Such studies are of considerable interest.

The locomotor response to amphetamine in the CN mutant mice was smaller
than that of controls when normalized to basal levels. However, it should be
noted that the absolute level of locomoter activity is higher in CN mutant
mice than controls after amphetamine administration.

Supportive Evidence. As CN is a key molecule in the immune system
(3), the CN inhibitors,
cyclosporin A and FK-506, are widely used as immunosuppressants. Psychotropic
effects of these compounds including agitation, restlessness, anxiety,
insomnia, confusion, visual/auditory hallucination, delusion, paranoia,
depression, apathy, and flattened affect have been reported
(30–32).
In addition, the lower incidence of rheumatoid arthritis in schizophrenic
patients (33) and a variety of
comorbidities, including diabetes
(34) and cardiovascular
problems (35), are consistent
with therapeutic (36) or side
effects of CN inhibitors (3,
37,
38). Moreover, various immune
abnormalities observed in schizophrenia, particularly alterations in levels of
cytokines such as Il-2, are consistent with altered CN signaling
(39,
40).

Recently, DNA microarray analysis was used to assay gene expression levels
in postmortem brains of schizophrenic patients
(41,
42). Expression of 10 genes
with functions known to be related to CN was found to be altered (see
Supporting Text for details). In addition, transcripts encoding
proteins involved in the regulation of presynaptic function have been found to
be decreased in postmortem brains of schizophrenic patients
(41). CN is critically
involved in presynaptic function of neurons
(43–45).
Caution is required in interpreting such studies as schizophrenic patients are
often subject to chronic medication with possible effects on gene expression.
In this regard, a recent study showed that administration of the
antipsychotic, clozapine, induced an increased expression of the CNA gene in
the prefrontal cortex (46) of
rats. Clozapine and haloperidol have also been found to increase IL-2
induction (47), suggesting
their possible activating effects on the CN signaling pathway. Our findings
raise the possibility that antipsychotic drugs may exert their effects, at
least in part, by modulating CN-signaling.

CN Genes and Schizophrenia Susceptibility. Based on the behavioral
abnormalities observed in CN mutant mice, we have directly investigated the
possibility that alterations in CN signaling could contribute to schizophrenia
susceptibility. In the accompanying article
(8), we report our initial
analysis of potential association of genes encoding CN subunits and
CN-interacting molecules with schizophrenia and provide direct genetic
evidence supporting the association of the PPP3CC gene encoding the CNAγ
catalytic subunit with disease.

Conclusion

We have assembled several lines of evidence indicating that altered CN
signaling could comprise an important contributing factor in schizophrenia
pathogenesis. Further analysis of the various pathways downstream of CN
activity is of considerable interest. Application of conditional gene
targeting technology should be particularly useful for this line of
investigation. We have demonstrated that the strategy of using a comprehensive
behavioral test battery on genetically engineered mice is a useful tool to
identify schizophrenia susceptibility genes.

Acknowledgments

We thank Hiroki Hamanaka for his technical assistance, Takeshi Yagi for
helping T.M. to develop home cage social interaction analysis system, and
Junji Ichikawa and Akinori Nishi for insightful discussion. This research was
supported by funding from National Institutes of Health Grant MH58880-03 (to
S.T.), the Howard Hughes Medical Institute (S.T.), RIKEN (S.T.), and a
National Alliance for Research on Schizophrenia and Depression Young
Investigator Award (to T.M.).

Footnotes

↵¶
To whom correspondence should be addressed. E-mail:
tonegawa{at}mit.edu.

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